Prior to the introduction of live attenuated mumps vaccines, mumps virus (MuV) was the leading cause of aseptic meningitis. Mumps incidence declined sharply due to vaccine use, although the recent global resurgence highlights the importance of maintaining robust vaccination programs. Even though it has been over 75 years since MuV was identified as the etiological agent of the disease, virtually nothing is known about the basis of virus neurotropism and the molecular determinants governing attenuation have not been identified. This complicates the development of safe vaccines as demonstrated by the fact that some licensed mumps vaccines caused aseptic meningitis. Mumps has recently reemerged in the US and Europe causing large outbreaks in vaccinated individuals. Since new MuV vaccines are currently in the pipeline this makes research to advance vaccine safety a priority. Our goal is to develop a toolkit of validated, robust in vitro, ex vivo and in vivo assays which predict whether or not mumps vaccines are safe for use in humans. The program of work has both immediate and long term importance for the field of vaccinology and the data will inform safety testing of rationally-attenuated MuV vaccines which are currently in development. Three synergistic and sequential aims are proposed: 1. Determine the primary cells, tissues and organs targeted by wild-type MuV in vivo: Recombinant (r) wild-type viruses of known provenance will be used to assess replicative capacity and tropism in disease- relevant primary cells, tissues and a rodent model of neurovirulence. We will test the model that MuV initially infects epithelia in the upper respiratory tract by infecting rhesus macaques with an rMV-expressing enhanced green fluorescent protein to answer the question, what are the primary target sites of MuV replication in vivo? 2. Determine if the primary cells targeted by highly attenuated and under-attenuated MuVs differ from each other and from the currently circulating wild-type virus: Understanding differences and similarities between safe and unsafe vaccines is critical for the development and licensing of safe vaccines. Enhanced green fluorescent protein-expressing MuVs derived from highly attenuated and under-attenuated strains will be assessed in the in vitro, ex vivo and in vivo models to determine if virus replication, tissue tropism and dissemination in vivo correlate with the known safety profile in human vaccinees. 3. Develop in vivo predictors for mumps vaccine safety using non-recombinant vaccine progenitors and rationally attenuated rMuVs which have differing replicative capacity and tropism: A set of vaccine progenitors and rMuVs with gene replacements and targeted mutations designed to modulate replicative capacity and tropism will be characterized. Based on their attenuation signature macaques will be vaccinated with the rMuVs and the safety profile will be assessed. This will answer the question can we develop robust in vitro, ex vivo and in vivo models which predict the likelihood that MuV vaccines will be safe in humans.